The glycine receptor is a member of the Cys-loop, ligand-gated ion channel family and is responsible for inhibition in the CNS. We examined the orientation of amino acids I229 in transmembrane 1 (TM1) and A288 in TM3, which are both critical for alcohol and volatile anesthetic action. We mutated these two amino acids to cysteines either singly or in double mutants and expressed the receptors in Xenopus laevis oocytes. We tested whether disulfide bonds could form between A288C in TM3 paired with M227C, Y228C, I229C, or S231C in TM1. Application of cross-linking (mercuric chloride) or oxidizing (iodine) agents had no significant effect on the glycine response of wild-type receptors or the single mutants. In contrast, the glycine response of the I229C/A288C double mutant was diminished after application of either mercuric chloride or iodine only in the presence of glycine, indicating that channel gating causes I229C and A288C to fluctuate to be within 6 A apart and form a disulfide bond. Molecular modeling was used to thread the glycine receptor sequence onto a nicotinic acetylcholine receptor template, further demonstrating that I229 and A288 are near-neighbors that can cross-link and providing evidence that these residues contribute to a single binding cavity.